RT-PCR is the technique of choice for analyzing
mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection
combines the ease and necessary exactness to be able to produce reliable
as well as rapid results. To obtain high accuracy and reliability
in RT and real-time PCR highly defined quantification strategies and calculation
methods are needed.

The reverse transcription - polymerase
chain reaction (RT-PCR) is the most sensitive method for the detection
of low abundance mRNA, often obtained from limited tissue samples. However,
real-time RT-PCR is a complex technique, there are substantial problems
associated with its true sensitivity, reproducibility and specificity and,
as a quantitative method, it suffers from the problems inherent in RT and
PCR. The recent introduction of fluorescence based kinetic (real-time)
RT-PCR procedures significantly simplifies the process of producing reproducible
quantification of mRNAs and promises to overcome these limitations. Nevertheless,
their successful application depends on a clear understanding of the practical
problems, and careful experimental design, application and validation remain
essential for accurate quantitative measurements of the transcriptom.